Jan 24, 2024
Protocol for preparing post-mortem tissue for intracellular neuromelanin quantification in H&E-stained sections.
- Marta Gonzalez-Sepulveda1,2,
- Thais Cuadros1,2,
- Miquel Vila1,3,4,5
- 1Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute (VHIR)-Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), 08035 Barcelona, Spain;
- 22Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 4Institute of Neurosciences, Autonomous University of Barcelona (INc-UAB), Bellaterra, Barcelona, Spain;
- 5Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain
- Vilalab Public
- Nuria
External link: https://doi.org/10.1038/s41467-024-53168-7
Protocol Citation: Marta Gonzalez-Sepulveda, Thais Cuadros, Miquel Vila 2024. Protocol for preparing post-mortem tissue for intracellular neuromelanin quantification in H&E-stained sections.. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pk1qgwz/v1
Manuscript citation:
Laguna, A., Peñuelas, N., Gonzalez-Sepulveda, M. et al. Modelling human neuronal catecholaminergic pigmentation in rodents recapitulates age-related neurodegenerative deficits. Nat Commun 15, 8819 (2024). https://doi.org/10.1038/s41467-024-53168-7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2024
Last Modified: January 24, 2024
Protocol Integer ID: 94079
Keywords: intracellular neuromelanin quantification, stained section
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020505
Abstract
Protocol for preparing post-mortem tissue for intracellular neuromelanin quantification in H&E-stained sections
Troubleshooting
Tissue Processing
Formalin-fixed paraffin-embedded tissue blocks from the pontine and midbrain regions were sectioned at 5µm, collected onto adhesive microscope slides and allowed to dry at room temperature for 24 hours.
Slides were incubated in the oven at 60°C for 30 minutes to melt the paraffin.
To remove the paraffin, sections were submerged in Xylene (Panreac Applichem 211769.2714) for 3 x 3 minutes, followed by rehydration in decreasing ethanol concentrations (100% ethanol for 2 x 5 minutes, 95% ethanol for 2 x 5, 70% ethanol for 2x5 minutes) and distilled H2O for 5 minutes.
Standard hematoxylin-eosin (H&E) staining.
Image Acquisition
Sections were scanned using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal neuromelanin signal quality with an automated Slide Scanner Olympus (SLIDEVIEW VS200, Tokyo, Japan).
Identical parameters were applied to each scanned section to ensure consistency in capturing neuromelanin.